Agave spirits have gained global recognition and hold a central position within the cultural heritage of Mexico. Traditional distilleries characterized by open fermentations driven by local microbial communities, persist despite the rise of industrial-scale counterparts. In this review, we explore the environmental conditions and production practices that make the must of cooked agave stems a unique habitat for colonizing microorganisms. Additionally, we review selected studies that have characterized yeast species within these communities, with a focus on their metabolic traits and genomic features. Over fifty fungal species, predominantly Saccharomycetales and few Basidiomycetes, along with a similar number of lactic and acetic acid bacteria, have been identified. Despite variations in the chemical composition of the agave substrate and diversity of cultural practices associated with each traditional fermentation process, yeast species such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Torulaspora delbrueckii, and several Pichia species have been consistently isolated across all agave spirit-producing regions. Importantly, cooked agave must is rich in fermentable sugars, yet it also contains inhibitory compounds that influence the proliferation dynamics of the microbial community. We discuss some of the genetic traits that may enable yeasts to flourish in this challenging environment and how human practices may shape microbial diversity by promoting the selection of microbes that are well-adapted to agave fermentation environments. The increasing demand for agave spirits, combined with concerns about the preservation of natural resources and cultural practices associated with their production, underscores the need to deepen our understanding of all key players, including the yeast communities involved.